The overall goal of this Program Project Grant is to define some of the fundamental mechanisms regulating distribution of nutritive blood flow to the brain. In this regard, studies within Project 1 will focus on the molecular, cellular and signal transduction events mediating autoregulation of nutritive cerebral blood flow (CBF) in response to step elevations in transmural pressure. We have recently cloned and sequenced a P4504A omega-hydroxylase cDNA within isolated pre-capillary arteriolar muscle cells which expresses an enzyme catalyzing formation of 20-hydroxyeicosetrinoic acid (20-HETE) from arachidonic acid (AA). Inhibition of omega-hydroxylases abolishes the normal, nearly perfect, autoregulation of laser-Doppler measured blood flow recorded via a cranial window in the rat parietal cortex. We have demonstrated that 20- HETE IS endogenously produced in arteriolar muscle where it potently ( (<10/-10M) inhibits activity of the large conductance Ca/2+ activated K+ channel (K/Ca), depolarizes the plasma membrane increases [Ca/2+]i and activates contractile elements. Preliminary findings show that 20-HETE also directly enhances inward L-type Ca/2+ channel current in patch- clamped arteriolar muscle cells. The signal transduction cascade mediating the contractile action of 20-HETE involves activation of PKC as evidenced by inhibition of its action in the presence of a PKC pseudosubstrate inhibitor Myr psiPKC-I(19-27). In addition, we have preliminary data showing that phosphorylation of myristolylated alanine- rich C kinase substrate (MARCKS) occurs in a 20-HETE dependent manner in primary cultures of cerebral arterial muscle cells. Protocols within Project 1 will focus on further defining the molecular expression of P4504A within the cerebral microcirculation, localization of P4504A omega-hydroxylase isoforms in the brain, the physiological significance of P4504A over-expression, and up-regulation of the AA omega-hydroxylase enzyme by regulating substrate availability. The substrate we will focus on will be molecular oxygen. We have recently demonstrated that the P4504A omega-hydroxylase possesses a Km for oxygen of approximately 60 torr. As PO/2 falls from 100 to 200 torr, there is a linear reduction of 20-HETE formation suggesting that P4504A enzymes may function as an oxygen sensory in the brain. We will define cerebral autoregulatory capacity under hypoxic and hyperoxic conditions. The studies as outlined in this Project relate to and are closely integrated into the other Projects of this Program. This Project, and the Program as a whole will significantly advance our knowledge regarding the molecular mechanisms regulating pre-capillary arteriolar caliber and how these mechanism s relate to the distribution of blood flow to the brain in the intact animal.
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